Cell-Mediated Immunity
The vertebrate immune system is constantly active against invading microbes and malignant cells. It is well known that the adaptive immune system shows a much stronger response on second, as compared to first, encounter with an antigen. This fact is exploited in vaccination, which works by inducing a state of lasting immunity known as immunological memory. Immunological memory requires the activation of T lymphocytes, specific for the infectious agent. T lymphocytes detect infection within cells by recognizing--via the T-cell receptor (TCR)--peptide fragments derived from the pathogen. However, most T lymphocytes are "MHC restricted", i.e. they recognize only complexes of peptides bound to highly polymorphic membrane proteins, encoded by class I and class II genes of the major histocompatibility complex (MHC) and presented on the surface on an accessory cell (designated an antigen-presenting cell or APC), in which the antigen has been processed. T lymphocytes can be classified as CD4.sup.+ or CD8.sup.+, depending on the specificity of an adherence receptor molecule. The CD4 adherence receptor recognizes MHC class II molecules, while CD8 binds class I. In addition, MHC restriction is further dependent on direct binding of the MHC molecule to certain parts of the TCR (Jorgensen et al., 1992).
CD4.sup.+ T cells (Helper T cells) activate macrophages and antibody-producing B cells, while CD8.sup.+ T cells (Cytotoxic T cells, CTL) kill cells infected by viruses and intracellular bacteria. Antigens can be processed by one of two pathways, depending on their origin. In the first pathway foreign material from outside the cell is engulfed by a specialized antigen-presenting cell (often a macrophage or B-cell), which breaks down the material and links the processed antigen to class II MHC molecules. The complexes are transported to the cell surface and presented to Helper T cells. The second pathway is generally concerned with processing of proteins made within virus-infected or malignant cells. These proteins are processed in the cells, i.e. they are subjected to partial proteolysis so as to form peptide fragments. These fragments then associate with class I MHC molecules and are transported to the cell surface for presentation to Cytotoxic T cells.
The processing of antigens by separate pathways makes biological sense. Thus antigens taken up from the surroundings eventually elicit B cells to produce antibodies which will be capable of protecting the organism against a subsequent challenge by the exogenous antigen. On the other hand, in the case of antigens in the form of abnormal structures made within an abnormal or errant cell (for example a virus-infected or malignant cell), it is advantageous for the immune system to be activated with a result leading eventually to the killing of the errant cell.
MHC-Bindin Peptides
In recent years there has been considerable progress in the analysis of peptides bound to MHC class I and II molecules (For reviews see e.g. Janeway, 1991; Rotzschke & Falk, 1991; Stauss, 1991; Tsomides & Eisen, 1991). Thus it has been found that MHC class I molecules bind short peptides of only about 8-12 amino acids and that MHC class II molecules bind peptides of about 10-17 amino acids.
Furthermore it has been found that the peptide fragments resulting from processing of antigens are transported to the cell surface bound in a groove on the extracellular part of an MHC molecule. For MHC class I molecules it has been found that individual amino acid side-chains, located at precise positions along the peptide, bind into the peptide-binding groove (Madden et at., 1991). The position of these pockets and the amino acids that line them can be different for different allelic variants. Consequently, different MHC class I molecule can bind a different set of peptides, and allele-specific motifs have been found for various MHC alleles (Van Bleek & Nathenson, 1990; Falk et at., 1991; Jardetzky et al., 1991). For example, peptides binding HLA-A2.1 preferably have L or M in position 2 and V or L in position 9, i.e. in the C-terminal position (Rotzschke & Falk, 1991).
Examples of peptides capable of binding MHC class I molecules in the murine system are A S N E N M E T M and S G P S N T P P E I, (SEQ ID NOS: 1 and 2), which both are presented by H-2-D.sup.b molecules. An example of a peptide capable of binding an MCH class I molecule in the human system is G I L G F V F T L, (SEQ ID NO: 3), which is presented by HLA-A2.1 molecules (Falk et al., 1991).
Conjugation of a MHC (class II) binding peptide, D Y G I L Q I N S R, (SEQ ID NO: 19), to a carbohydrate, 4-O-.alpha.-D-galactopyranosyl-.beta.D-galactopyranose, is described in Elofsson et at (1991). WO 89/07448 discloses compositions for modulating the immune response of a host, employing peptides having homology with peptides binding MHC class I molecules.
Synthetic Peptides as Carriers for T Cell Specific Epitopes
CTL:s can be generated from spleens of mice painted with trinitrochlorobenzene (TNP), and selected for killing of TNP-coated syngenic target cells. Some CTL:s generated like this have been shown to recognize short MHC class I (K.sup.b)-bound peptides of 10 amino acids with TNP bound to an internal (position 6) lysine residue. Furthermore, some CTL:s kill syngenic target cells that have processed and presented TNP bound to different carrier proteins (MSA, BSA and KLH) and also TNP-coated, allogenic target cells (Ortmann et al., 1992)
Tumor-Associated Antigens
The possibility that tumors may be recognized as foreign by the immune system (based on the abnormal character of tumor cells) would offer valuable opportunity for developing effective cancer therapies. In experimental systems tumors have shown to be highly immunogenic and the triggered immune responses was sufficient to eliminate the tumor cells. In the clinical situation, however, a tumor, being a product of multiple genetic and adaptive alterations, has little immunogenicity at the time when it becomes a medical problem. Accordingly, few truly tumor associated protein antigens, i.e. antigens being expressed in tumor cells only, have been found.
In contrast to the lack of tumor associated protein antigens, a variety of aberrant carbohydrate (CHO) structures are present on tumor cells (for a review see Hakomori, 1991). These are formed as a consequence of abnormalities in the enzyme systems responsible for the assembly of the CHO chains. The chains are often truncated with the result that CHO epitopes (which are absent or hidden on normal cells) are expressed on tumor cells.
Aberrant CHO structures on tumor cells can exist in the form of glycoprotein, glycolipid or a complex of both of these two forms. Glycoproteins are often secreted into body fluids whereas glycolipids to a large extent are membrane bound.
Examples of tumor associated carbohydrate antigens are the GM3 ganglioside, which has been identified in mouse melanoma B16 cells (Nores et al., 1987), GD3 ganglioside associated with human melanoma cells (Portoukalian et al., 1979) and Gb3 ganglioside which is expressed in Burkitt lymphoma cells (Wiels et at, 1981; Brodin et al., 1988).
Carbohydrates Associated with Infectious Diseases
Carbohydrates associated with infectious diseases can be expressed on the infectious agents, on secreted or shed material from these, or on the surface of infected host cells.
There are some examples of verified, or putative, carbohydrate-specific T cell responses, associated with diseases caused by infectious agents like Salmonella typhimudum (Robertsson et al., 1982), Leishmania major (Moll et al., 1989), Candida albicans (Domer et al., 1989) and Mycobacteria (Crowle, 1988).
Carbohydrate antigens associated with HIV infection are described in Hansen et at (1990).
Immunotherapy Against Tumors
Immunotherapy against tumors has a long medical history and has been tried by both non-specific and specific means. In non-specific manipulations, the immune system has been activated by various factors such as BCG, IL-2 etc., in order to increase the existing background immunity against the tumor. In specific manipulations, vaccines have been constructed using tumor cells or material derived from these.
T cell-mediated immune responses play crucial roles in antitumor immunity. Whereas the mechanisms of activation of T cell responses against proteins are characterized to some extent (see above), they are essentially unknown for carbohydrates. Carbohydrate antigens are poor candidates for presentation to MHC molecules, because of structural constraints imposed by the peptide-binding groove of the MHC proteins. Nevertheless, Ishioka et al. (1992) has shown that the CHO moiety can be an important part of the antigenic determinant recognized by T cells, and suggest that although there are limitations to the generation of CHO-specific T cell responses, such responses could be generated by judicious placement of the carbohydrate within a MHC-binding peptide.
Longenecker and coworkers (WO 88/00053; Henningson et al., 1987) describe the use of "Synthetic Tumor-Associated Glycoconjugates" (S-TAGs) for stimulation of anticancer T cell immunity. In these experiments, synthetic Thomsen-Friedenreich (TF) and Tn antigens, carbohydrates expressed on most human adenocarcinomas, were conjugated to a carrier protein and used to demonstrate that delayed-type hypersensitivity (DTH) effector cells could recognize and respond to the carbohydrate determinants. An S-TAG composed of TF antigen coupled to carrier (keyhole limpet hemocyanin) and emulsified in Ribi adjuvant was administered to mice with TA3-Ha adenocarcinoma. When administration was preceded by treatment with cyclophosphamide, a 50-90% long term survival of the hosts was observed (Fung et al., 1990).
Thurin et al. (1991) describe a conjugate comprising (i) a tumor-associated carbohydrate hapten; (ii) a peptide containing a viral mouse Helper T cell epitope; and (iii) a Quil-A glycoside based adjuvant. Mice immunized with the conjugate showed 1 gM responses specific for the hapten together with a Helper T cell reactivity to the viral epitope.